This case report presents a successful case of autologous tooth transplantation assisted by three-dimensional (3D) printing technology combined with delayed partial pulpotomy. Preoperative cone-beam computed tomography (CBCT) data of the patient were collected, and Mimics software was used to design and construct a 3D template for the transplanted tooth. After disinfection, the template was used to guide the preparation of the alveolar socket at the recipient site. Following the extraction of the hopeless tooth, the recipient socket was prepared with the aid of the template. 3D printing-assisted digital surgery enabled accurate preparation of the recipient site, reduced the extraoral time of the transplanted tooth, and preserved the viability of the periodontal membrane. The physical dimension of the apical foramen is a critical determinant of revascularization of the transplanted dental pulp. In this case, the apical foramen diameters of the mesial and distal roots were 0.68 mm and 0.91 mm, respectively. Postoperatively, vital pulp preservation was achieved in the distal root with a larger apical foramen. This case provides a novel clinical reference for vital pulp preservation during mature autologous permanent tooth transplantation.